Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells

Here we report on the feasibility of using replication deficient adenoviruses to modify signal transduction systems in epithelia. We constructed two viruses, one expressing a dominant negative mutant of the alpha-subunit of G(q) (Ad-EF1-dnG alpha(q)) and the other expressing the wild-type alpha-subunit of G(q) (Ad-EF1-wtG alpha(q)). We used an adenovirus expressing green fluorescent protein (Ad-EF1-GFP20) to show that infection of cultured cells with an adenovirus results in at least 95% expression of the transgene in both HSG and HT29 cells. We also used an adenovirus that expresses no transgene (Ad-MX17) to demonstrate that adenoviral infection itself does not affect the resting concentration of cytosolic Ca2+ ([Ca2+](i)) or the carbachol responses in these cells. We further show that Ad-EF1-dnG alpha(q) inhibits the increase in [Ca2+](i) produced by muscarinic receptor activation in both the cell lines we studied. This inhibitory effect is not shared by Ad-EF1-wtG alpha(q), which indicates that in both HSG and HT29 cells, the increase in [Ca2+](i) produced by muscarinic receptor activation is largely mediated by activation of G(q). Neither virus affected the resting level of [Ca2+](i) in these cells. Our findings confirm the feasibility of using replication deficient adenoviruses expressing dominant negative mutants to investigate the role of G proteins in signal transduction systems.